uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line

Urokinase plasminogen activator receptor （uPAR） plays a major role in cancer-invasion and metastasis and uPAR expression is correlated with a poor prognosis in various cancer types. Moreover, the expression of uPAR is increased under hypoxic conditions. Nitric oxide （NO） and its metabolites produced by inducible nitric oxide synthase （iNOS） are important products ofhypoxic stress, and NO may activate or modulate extracellular signal regulated kinase （ERK）. Here, we evaluated uPA, uPAR, and activated ERK levels under hypoxic conditions, and the modulatory effects of iNOS and NO in the MDA-MB-231 human breast cancer cell line. Cells were incubated in a hypoxic or normoxic incubator and treated with PD98059 （a MEK 1/2 inhibitor, which abrogates ERK phosphorylation） and aminoguanidine （a selective iNOS inhibitor）, uPAR expression, ERK phosphorylation, and uPA activity were found to be increased under hypoxic conditions. Moreover, when cells were treated with PD98059 under hypoxic conditions, uPAR was downregulated, whereas aminoguanidine markedly increased ERK phosphorylation in a dose dependent manner. Furthermore, aminoguanidine increased uPAR expression and prevented the inhibition of uPAR expression by PD98059. These results demonstrated that uPAR is induced by hypoxia and that increased uPAR expression is mediated by ERK phosphorylation, which in turn is modulated by iNOS/NO in MDA-MB-231 cells. We conclude that iNOS/NO downregulates the expression of uPAR under hypoxic conditions via ERK pathway modulation.     

Detergent-free caveolae proteome suggests an interaction with ER and mitochondria

Recent proteomic studies of detergent resistant membrane fractions have begun to characterize the protein composition of caveolae and lipid rafts. The methods used in most of these studies, however, are not able to distinguish between plasma membrane and internal membrane lipid domains. Here we used a non-detergent method for obtaining fractions enriched in caveolae derived from the plasma membrane of multiple cell types. Unexpectedly, the proteins in the caveolae proteome suggest these lipid domains may interact with elements of ER and mitochondria. A comparison of the partial proteome we obtained with other published reports identifies 26 proteins that are candidate marker proteins for identifying caveolae in multiple cell types.     

Higenamine reduces apoptotic cell death by induction of heme oxygenase-1 in rat myocardial ischemia-reperfusion injury

Pharmacological modulation of heme oxygenase (HO) gene expression may have significant therapeutic potential in oxidant-induced disorders, such as ischemia reperfusion (I/R) injury. Higenamine is known to reduce ischemic damages by unknown mechanism(s). The protective effect of higenamine on myocardial I/R-induced injury was investigated. Ligation of rat left anterior descending coronary artery for 30 min under anesthesia was done and followed by 24 h reperfusion before sacrifice. I/R-induced myocardial damages were associated with mitochondria-dependent apoptosis as evidenced by the increase of cytochrome c release and caspase-3 activity. Administration of higenamine (bolus, i.p) 1 h prior to I/R-injury significantly decreased the release of cytochrome c, caspase-3 activity, and Bax expression but up-regulated the expression of Bcl-2, HO-1, and HO enzyme activity in the left ventricles, which were inhibited by ZnPP IX, an enzyme inhibitor of HO-1. In addition, DNA-strand break-, immunohistochemical-analysis, and TUNEL staining also supported the anti-apoptotic effect of higenamine in I/R-injury. Most importantly, administration of ZnPP IX inhibited the beneficial effect of higenamine. Taken together, it is concluded that HO-1 plays a core role for the protective action of higenamine in I/R-induced myocardial injury.     

Extreme regression

We develop a new method for describing patient characteristics associated with extreme good or poor outcome. We address the problem with a regression model composed of extrema (maximum and minimum) functions of the predictor variables. This class of models allows for simple regression function inversion and results in level sets of the regression function which can be expressed as interpretable Boolean combinations of decisions based on individual predictors. We develop an estimation algorithm and present clinical applications to symptoms data for patients with Hodgkin's disease and survival data for patients with multiple myeloma.     

Classification methods for the development of genomic signatures from high-dimensional data

Personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies. We present Classification by Ensembles from Random Partitions (CERP) for class prediction and apply CERP to genomic data on leukemia patients and to genomic data with several clinical variables on breast cancer patients. CERP performs consistently well compared to the other classification algorithms. The predictive accuracy can be improved by adding some relevant clinical/histopathological measurements to the genomic data.     

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.     

Expression, purification and transduction of PEP-1-botulinum neurotoxin type A (PEP-1-BoNT/A) into skin

Botulinum neurotoxin A (BoNT/A) has been used therapeutically to treat muscular hypercontractions and sudomotor hyperactivity and it has been reported that BoNT/A might have analgesic properties in headache. PEP-1 peptide is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In this study, a BoNT/A gene were fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-BoNT/A fusion protein. The expressed and purified PEP-1-BoNT/A fusion proteins were efficiently transduced into cells in a time- and dose-dependent manner when added exogenously in a culture medium. In addition, immunohistochemical analysis revealed that PEP-1-BoNT/A fusion protein efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin. These results suggest that PEP-1-BoNT/A fusion protein provide an efficient strategy for therapeutic delivery in various human diseases related to this protein.     

The development and validation of a charge-coupled device laser reflectance system to measure the complex cross-sectional shape and area of soft tissues

Determination of the stresses in soft tissues such as ligaments and tendons under uniaxial tension require accurate measurement of their cross-sectional area. Of the many methods available, there are concerns regarding contact methods which exert external loads and deform the cross-sectional shape of soft tissues. Hence, the area measurements are affected. On the other hand, non-contact methods have difficulties in dealing with complex shapes, especially with concavities. To address these problems, a new measurement system using a charge-coupled device (CCD) laser displacement sensor has been developed and tested. This system measures the complete surface profile of the object by rotating the laser 360° around the soft tissue. Then, the cross-sectional shape is reconstructed and the cross-sectional area determined via Simpson's rule. The system's accuracy was first verified with objects of various cross-sectional shapes and areas (cylinder: 23.1, 76.5, 510.3 mm²; cuboid: 34.3, 163.8, 316.7 mm², and cylinder with concavities: 121.4 mm²). The CCD laser reflectance system's accuracy was within 2.0% for these objects. To test biological application, the goat Achilles tendon and the anteromedial bundle of the porcine anterior cruciate ligament specimens were measured and compared to values obtained using another accepted technique, the laser micrometer system. The areas obtained using the CCD laser reflectance system were 4.4% and 9.7% lower than those obtained with the laser micrometer system respectively. These differences could be mainly attributed to concavities. Thus, the CCD laser reflectance system is an improved method for measuring the cross-sectional shape and area of soft tissues since it can detect and account for concavities without physically contacting the specimen.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.